RESUMEN
To determine the activity of all human Cdc25 phosphatases, two different methods are described. For assaying phosphatase activities of recombinant Cdc25 proteins produced in Escherichia coli or insect cells, a fluorimetric assay using fluorescein diphosphate (FDP) as a substrate is recommended. To analyze endogenous Cdc25 phosphatase activities of immunoprecipitates from total cellular extracts, the physiological substrate Cdk1/cyclin B1 is most sensitive.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Quinasas/metabolismo , Fosfatasas cdc25/análisis , Animales , Ciclina B/metabolismo , Ciclina B1 , Escherichia coli/metabolismo , Fluoresceína/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Fosforilación , Proteínas Recombinantes de Fusión/análisis , Fase S , Spodoptera/citología , Spodoptera/genéticaRESUMEN
The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclin E and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation, Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. We found that in response to UV irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclin E/Cdk2 kinase activity was high. Furthermore, we find that Cdc25A was phosphorylated by Chk1 on Ser-75 in vitro and that the same site was also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Serina/química , Fosfatasas cdc25/química , Alanina/química , Western Blotting , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Ciclina E/metabolismo , ADN/metabolismo , Daño del ADN , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/metabolismo , Células HeLa , Histidina/química , Humanos , Modelos Biológicos , Mutación , Mapeo Peptídico , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Fase S , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
In addition to inducing transcription of a battery of target genes encoding drug-metabolizing enzymes, the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce antiestrogenic responses. However, the mechanisms underlying such complex biologic responses affecting growth and differentiation remain unclear. In the present study we have investigated biological effects of a constitutively active mutant of the aryl hydrocarbon (Ah) receptor (CA-AhR), in particular whether it modulates estrogen receptor function in human MCF-7 breast cancer cells. To this end, the CA-AhR protein was conditionally expressed using the tet repressor. Expression of CA-AhR resulted in constitutive formation of a DNA-binding AhR-aryl hydrocarbon receptor nuclear translocator heterodimeric complex and enhanced expression of the Ah receptor target gene CYP1A1 in the absence of TCDD. Moreover, expression of CA-AhR inhibited estrogen-dependent cathepsin D expression and growth of these cells. Thus, the present model system conditionally expressing the CA-AhR protein provides a novel tool for the investigation of AhR-mediated signaling pathways.